Human immunodeficiency virus-1 (HIV-1) persists in the latent reservoir as transcriptionally silent and integrated proviruses. These integrated proviruses consist of three types: induced proviruses, intact noninduced proviruses and defective proviruses. A HIV-1 latency measurement assay which can distinguish inducible from defective proviruses is essential to evaluate the efficacy of eradication efforts. Quantitative HIV 1 DNA PCR assays measure all proviruses, the vast majority of which are defective. Thus, this assay overestimates the size of the latent reservoir. The quantitative viral outgrowth assay (Q-VOA) measures the induced proviruses; however, it underestimates the size of the reservoir because it does not detect the intact noninduced proviruses. These proviruses failed to produce detectable viral outgrowth after one round of maximum T cell activation but possess the full potential to be induced and produce replication-competent viruses upon subsequent stimulation. Quantitative reverse-transcription PCR assays for cell-associated HIV-1 RNA detection measure either HIV-1 RNA copy numbers or the frequency of HIV-1 infected cells induced to express HIV-1 RNA after latency reversal. However, the measurement of HIV-1 RNA copy numbers does not indicate the number of latently infected cells involved, while the measurement of frequency of HIV-1 infected cells requires labor-intensive limiting dilution culture. A simple and high-throughput assay to measure the frequency of patient cells containing inducible proviruses is critical to the HIV-1 cure effort. The presence of intact noninduced proviruses indicates that even maximum T cell activation fails to reactivate all inducible HIV-1 proviruses. We hypothesize that this is because the cellular environment is not permissive for all intact proviruses to become fully reactivated. To understand the cellular environment necessary for an intact provirus to be induced, an important approach is to compare the transcriptome profiles of patient cells which are in favorable and less favorable states for HIV-1 gene expression. However, there is no existing cellular or viral marker which can distinguish patient cells in different permissive states for such comparison. We develop a RNA-preserving, HIV-1-specific, flow cytometry-based, fluorescent in situ hybridization assay (HIV-1 RNA flow-FISH) to measure the size of the latent reservoir. The frequency of latently infected cells will be measured as the number of events (cells containing HIV-1 RNA after maximum T cell activation), and the abundance of HIV-1 RNA in each cell will be measured by fluorescence intensity. This RNA-preserving HIV-1 RNA detection assay will then be used to sort out patient cells in favorable and less favorable states for HIV-1 gene expression so that their transcriptomes can be compared. This simple, novel, high throughput and affordable assay would not only provide a clinically-feasible HIV-1 latent reservoir measurement but also help to answer why noninduced proviruses are not induced, and how 100% HIV-1 reactivation can be achieved.